While invaluable understanding has been gained from structures of insulin (and more recently IGF-I) in complex with fragments of the insulin receptor (IR)(1), the mechanism of ligand engagement with the receptor (or with the highly similar IGF-1R) still is not entirely understood. We took a fluorescence resonance energy transfer (FRET) approach to probe for sites of interaction between IGF-II and the IGF-1R using coumarin (Cou) fluorescent probes within the classical binding site 1 and in site 2 of IGF-II, and endogenous Trp residues of IGF-1R as the FRET partner. Insulin receptor structural change has previously been monitored by a change in Trp fluorescence, suggesting that Trp may lie in close proximity to the ligand binding pockets(2). Analysis of available insulin:IR structures (and more recently IGF-I:IR/IGF-1R structures) revealed that several Trp residues could act as potential FRET partners with IGF-II site 1 and site 2 residues. Thus Phe19Cou IGF-II and Phe28Cou IGF-II proteins, with substitutions in site 1 and site 2 respectively, were produced using convergent synthesis or recombinant methods to incorporate the non-canonical Cou amino acid. These fluorescent analogues bind with nanomolar affinities to the IGF-1R. We demonstrated both are good probes for investigating the binding interactions of IGF-II with the IGF-1R and the outcomes of the FRET analyses will be discussed.
References: 1. Ward et al. (2013) BioEssays 35(11):945-954. 2. Lee J, Pilch PF, Shoelson SE, & Scarlata SF (1997) Biochemistry 36(9):2701-2708.